In the presence of NADH and oxygen, extracts from Klebsiella aerogenes inactivate glutamine synthetase. Inhibitors of the respiratory chain, cyanide, azide, and a quinoline-N-oxide, stimulate the inactivation of glutamine synthetase. However, catalase blocks the inactivation, suggesting the participation of hydrogen peroxide in the inactivation. Cyanide and azide may also act by inhibiting catalase. The inactivation discriminates between the adenylylated and unadenylylated fractions of glutamine synthetase. Extracts from different growths of bacteria preferentially inactivated either unadenylylated (EO) or adenylylated (E12) glutamine synthetase from E. coli. The extract also preferentially inactivated the unadenylylated fraction of endogenous glutamine synthetase in one preparation tested. The inactivating extract can be separated into two fractions with Cibachrome-gel chromatography. Both are required for inactivating activity. Both have apparent molecular weights greater than 10,000, and both are heat-labile. Non-enzymatic inactivation of glutamine synthetase occurs rapidly in the presence of ascorbic acid. Catalase also prevents this ascorbic-acid-mediated inactivation.